Hundreds of thousands of human genomes are now being sequenced to characterize genetic variation and use this information to augment association mapping studies of complex disorders and other phenotypic traits 1-4 . Genetic variation is identified mainly by mapping short reads to the reference genome or by performing local assembly 2,5-7 . However, these approaches are biased against discovery of structural variants and variation in the more complex parts of the genome. Hence, large-scale de novo assembly is needed. Here we show that it is possible to construct excellent de novo assemblies from high-coverage sequencing with mate-pair libraries extending up to 20 kilobases. We report de novo assemblies of 150 individuals (50 trios) from the GenomeDenmark project. The quality of these assemblies is similar to those obtained using the more expensive long-read technology 4,8-13 . We use the assemblies to identify a rich set of structural variants including many novel insertions and demonstrate how this variant catalogue enables further deciphering of known association mapping signals. We leverage the assemblies to provide 100 completely resolved major histocompatibility complex haplotypes and to resolve major parts of the Y chromosome. Our study provides a regional reference genome that we expect will improve the power of future association mapping studies and hence pave the way for precision medicine initiatives, which now are being launched in many countries including Denmark.Using a combination of high-depth (average 78× ) Illumina pairedend and mate-pair libraries, we applied Allpaths-LG 14 to create de novo assemblies of high quality and coverage for each of the 150 individuals with a median scaffold N50 of ~ 21 megabases (Mb; maximum ~ 30 Mb) (Supplementary Table 1). The 100 largest scaffolds in each of the 140 best assemblies typically covered more than 75% (median 77%, Extended Data Fig. 1a) of the genome, with the largest scaffolds exceeding 110 Mb in size (Supplementary Table 1). To evaluate the accuracy of the assemblies, we subsequently aligned the scaffolds for each individual to the human reference genome (GRCh38) 15 . Figure 1 shows an example individual where the euchromatic part of each chromosome was almost completely covered by a few large scaffolds and in several cases scaffolds covered almost entire chromosome arms. Only rarely did we find that large scaffolds break and align to more than one chromosome (Extended Data Fig. 1b), suggesting that even the largest scaffolds are seldom chimaeric. We also compared our de novo assemblies with a published long-read assembly based on BioNano mapping and PacBio sequencing 16 . Extended Data Figs 2a and 3 show that this assembly was less complete than our assemblies, but with similar scaffold lengths. The long-read assembly had 5.38% missing data compared with our median of 4.25% (Extended Data Fig. 3a), but the missing data in our assemblies were found in smaller gaps (Extended Data Fig. 3b, c), and the median contig length was therefore much smaller th...

The peroxisome proliferator-activated receptor ␥ (PPAR␥) is a key regulator of terminal adipocyte differentiation. PPAR␦ is expressed in preadipocytes, but the importance of this PPAR subtype in adipogenesis has been a matter of debate. Here we present a critical evaluation of the role of PPAR␦ in adipocyte differentiation. We demonstrate that treatment of NIH-3T3 fibroblasts overexpressing PPAR␦ with standard adipogenic inducers led to induction of PPAR␥2 expression and terminal adipocyte differentiation in a manner that was strictly dependent on simultaneous administration of a PPAR␦ ligand and methylisobutylxanthine (MIX) or other cAMP elevating agents. We further show that ligands and MIX synergistically stimulated PPAR␦-mediated transactivation. In 3T3-L1 preadipocytes, simultaneous administration of a PPAR␦-selective ligand and MIX significantly enhanced the early expression of PPAR␥ and ALBP/aP2, but only modestly promoted terminal differentiation as determined by lipid accumulation. Finally, we provide evidence that synergistic activation of PPAR␦ promotes mitotic clonal expansion in 3T3-L1 cells with or without forced expression of PPAR␦. In conclusion, our results suggest that PPAR␦ may play a role in the proliferation of adipocyte precursor cells, whereas activation of endogenous PPAR␦ in 3T3-L1 cells appears to have only minor impact on the processes leading to terminal adipocyte differentiation.Adipocyte differentiation proceeds in a cascade-like manner by the sequential action of different classes of transcriptional regulators among which members of the CCAAT/enhancerbinding protein (C/EBP) 1 and the peroxisome proliferator-activated receptor (PPAR) families play crucial roles, and in a complex interdependent manner regulate clonal expansion, withdrawal from the cell cycle, and terminal differentiation (reviewed in Refs. 1-4). The PPAR family belongs to the superfamily of nuclear hormone receptors and comprises three subtypes, PPAR␣, PPAR␦ (also designated PPAR␤, FAAR, or NUC-1) and PPAR␥, the latter of which exists in two isoforms (5-8). The PPARs are ligand-activated transcription factors that bind as heterodimers with members of the retinoid X receptor (RXR) subfamily to PPAR response elements (PPREs) in the promoters/ enhancers of responsive genes. The PPARs are activated by a large variety of fatty acids and fatty acid metabolites, and direct binding of many of these activators to PPARs has been demonstrated (9 -12). Synthetic thiazolidinedione insulin-sensitizing antidiabetic drugs have been shown to be high affinity PPAR␥ ligands (13), and selective PPAR␦ ligands have recently been described (14 -16). Apart from ligands, the transactivation potential of PPAR␥ and PPAR␣ is regulated by phosphorylation (17-23) and by interaction with different families of coactivators and corepressors (24,25), and interaction with ligands and cofactors may in part be controlled by phosphorylation (26, 27).Activators of PPAR␥ promote adipocyte differentiation of preadipocytes and multipotent C3H10T 1/2 cells (1...

The peroxisome proliferator-activated receptors (PPARs) are transcription factors involved in fatty acid metabolism and energy homeostasis. The PPARs also play crucial roles in the control of cellular growth and differentiation. Especially, the recently emerged concept of ligand-dependent PPARgamma-mediated inhibition of cancer cell proliferation through induction of G(1)-phase arrest and differentiation is of clinical interest to cancer therapy. Tetradecylthioacetic acid (TTA) is a sulphur-substituted saturated fatty acid analog with unique biochemical properties. In this study, we investigated the effects of TTA-administration on cell proliferation in glioma cancer models. The rat glioma cell line BT4Cn, whether grown in culture or implanted in rats, expressed significant levels of PPARgamma and PPARdelta, with PPARgamma being the predominant PPAR subtype. In BT4Cn cells, TTA activated all PPAR subtypes in a dose-dependent manner. In cell culture experiments, the PPARgamma-selective ligand BRL49653 moderately inhibited growth of BT4Cn cells, whereas administration of TTA resulted in a marked growth inhibition. Administration of the PPARgamma-selective antagonist GW9662 abolished BRL49653-induced growth inhibition, but only marginally reduced the effect of TTA. TTA reduced tumor growth and increased the survival time of rats with implanted BT4Cn tumor. TTA-induced apoptosis in BT4Cn cells, and the administration of TTA led to cytochrome c release from mitochondria and increased the glutathione content in glioma cells. In conclusion, our results indicate that TTA inhibits proliferation of glioma cancer cells through both PPARgamma-dependent and PPARgamma-independent pathways, of which the latter appears to predominate.

Nuclear factor-kappaB (NF-kappaB) is an inducible nuclear transcription factor regulating a range of cellular processes. An imbalance of the DNA binding activity of NF-kappaB may, therefore, be part of the pathophysiological mechanisms in psoriasis. The purpose of this study was to determine the NF-kappaB DNA binding activity in psoriatic skin using three different kappaB sites and to determine how DNA binding activity was modulated by the anti-psoriatic drug calcipotriol. By electrophoretic mobility shift assay, we demonstrated that the NF-kappaB DNA binding to the p53 kappaB site was decreased, whereas the NF-kappaB DNA binding to the interleukin-8 (IL-8) kappaB site was increased in lesional psoriatic skin compared with non-lesional psoriatic skin. No regulation was seen on the NF-kappaB DNA binding to the major histocompatibility complex class I kappaB site. These changes were paralleled by a similar decrease in p53 expression and an increase in IL-8 expression in involved psoriatic skin compared with uninvolved skin as determined by quantitative RT-PCR. The alteration in NF-kappaB DNA binding activity was neither accompanied by any change in the expression of the inhibitor kappaB (IkappaB) kinases, IKKalpha, IKKbeta, and IKKgamma nor in the expression of the NF-kappaB inhibitor proteins, IkappaBalpha and IkappaBbeta. Immunofluorescence analysis revealed that p65 was sequestered in the cytoplasm of keratinocytes, whereas p50 exhibited a cytoplasmic as well as a nuclear localization. Interestingly, this distribution of p50 and p65 was similar in lesional and non-lesional psoriatic skin. Topical application of calcipotriol to lesional psoriatic skin for 4 d resulted in increased NF-kappaB binding to the p53 kappaB site and decreased NF-kappaB binding to the IL-8 kappaB site. Taken together, our data demonstrate that the NF-kappaB DNA binding activity is regulated in a specific manner in psoriatic skin depending on the kappaB sites investigated, and that topical treatment of psoriatic skin normalizes the abnormal NF-kappaB binding activity seen in lesional psoriatic skin.

Peroxisome proliferator-activated receptors (PPARs) are important targets for drugs used in the treatment of atherosclerosis, dyslipidaemia, obesity, type 2 diabetes, and other diseases caused by abnormal regulation of the glucose and lipid metabolism. We applied a virtual screening workflow based on a combination of pharmacophore modeling with 3D shape and electrostatic similarity screening techniques to discover novel scaffolds for PPAR ligands. From the resulting 10 virtual screening hits, five tested positive in human PPAR ligand-binding domain (hPPAR-LBD) transactivation assays and showed affinities for PPAR in a competitive binding assay. Compounds 5, 7, and 8 were identified as PPAR-alpha agonists, whereas compounds 2 and 9 showed agonistic activity for hPPAR-gamma. Moreover, compound 9 was identified as a PPAR-delta antagonist. These results demonstrate that our virtual screening protocol is able to enrich novel scaffolds for PPAR ligands that could be useful for drug development in the area of atherosclerosis, dyslipidaemia, and type 2 diabetes.